Nicotine acetylcholine receptors (nAChRs) are membrane proteins that are prevalent in animal kingdom and have important physiological actions and clinical research significance, and they mediate many physiological functions of central and peripheral nervous systems, including learning, memory, response, analgesia and motion control. The nAChRs inactivate release of many neurotransmitters such as dopamine, noradrenaline, serotonin, γ-aminobutyric acid. It is confirmed that nAChRs are critical targets for screening medicines in diagnosis and treatment of a large group of important diseases, and these diseases include difficult miscellaneous diseases such as addiction, pains, cancers, amentia, Parkinson's disease, mental diseases, depression, myasthenia gravis. So far, there is no medicine for symptomatic treatment of these diseases. Common non-selective nAChR agonists such as nicotine could relieve symptoms of the above nerve diseases, but they have strong side-effects on heart and gastrointestinal tract and addiction. Hence, the key for treatment of the above diseases is to develop ligand medicines having high selectivity on various subtypes of nAChRs (Livett B G, Sandall D W, Keays D, Down J, Gayler K R, Satkunanathan N, Khalil Z. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon. 2006, 48(7):810-829. Nicke, A., Wonnacott, S. & Lewis, R. J. Alpha-conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. European journal of biochemistry/FEBS 271, 2004, 2305-2319. Dani, J. A. & Bertrand, D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annual review of pharmacology and toxicology 2007, 47:699-729).
However, the precondition for developing such medicines is to obtain selective compounds capable of specifically binding various subtypes of nAChRs, which could be directly used as therapeutic medicines for relevant diseases, or as tool medicines for studying and identifying fine compositions and physiological functions of the various subtypes. In addition, in tissues of breast cancer and lung cancer, the activation of nicotine acetylcholine receptors on tumor cytomembrane could be blocked with medicines so as to effectively conduct early diagnosis or treatment of these catastrophic cancers.
The nAChRs have many subtypes assembled with different α and β subunits, and each subtype has distinct pharmacological features. Due to the lack of highly selective ligand compounds for various subtypes, many difficulties should be overcome in studying and illustrating fine structures and functions of various nAChRs subtypes.
Studies show that α9α10 nAChR is a new target of medicine for treatment of neuropathic pain (McIntosh, J. M.; Absalom, N.; Chebib, M.; Elgoyhen, A. B.; Vincler, M., Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochemical pharmacology 2009, 78 (7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain research 2005, 1059 (2), 149-58.). The α9α10 nAChR blocking agent has function of alleviating neuropathic pain and accelerating recovery of injured neurons (Holtman, J. R.; Dwoskin, L. P.; Dowell, C.; Wala, E. P.; Zhang, Z.; Crooks, P. A.; McIntosh, J. M., The novel small molecule alpha9alpha10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic. European journal of pharmacology 2011, 670 (2-3), 500-8. Zheng, G.; Zhang, Z.; Dowell, C.; Wala, E.; Dwoskin, L. P.; Holtman, J. R.; McIntosh, J. M.; Crooks, P. A., Discovery of non-peptide, small molecule antagonists of alpha9alpha10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain. Bioorganic & medicinal chemistry letters 2011, 21 (8), 2476-9). The α9α10 nAChR of keratinocyte plays an important role in pathological physiology process of wound healing (Chernyaysky, A. I.; Arredondo, J.; Vetter, D. E.; Grando, S. A., Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Experimental cell research 2007, 313 (16), 3542-55). Recent studies show that α9 nAChR subunit is overexpressed in breast cancer tissues. Variant of α9 subunit affects transformation and proliferation of bronchial cells, so the subunit has very important significance in treatment of lung cancer (Chikova, A.; Grando, S. A., Naturally occurring variants of human Alpha9 nicotinic receptor differentially affect bronchial cell proliferation and transformation. PloS one 2011, 6 (11), e27978.).
Surveys show pains including arthritis, neuralgia and sore pain affect ⅙ of population, among which neuralgia affects 4-8% of population. Existing methods for treatment of neuralgia mainly involve in local anaesthesia medication to block pain signal generated by peripheral nerves, nerve plexus, dorsal root nerves and sympathetic nervous system. However, these treatments merely have short-term analgesic effect, and cannot permanently control neuralgia. Many diseases may induce neuralgia, including cancers and chemotherapy of cancers, alcoholism, ischialgia, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, myeloma, multipoint neuralgia, chronic congenital esthesioneurosis, acute fierce spontaneous neuralgia, squeezing neuralgia, angiitis (vasculitis)/ischemia, uremia, children biliary liver disease, chronic respiratory disorder, complex neuralgia, multiple organ failure, sepsis/pyaemia, hepatitis, porphyria, avitaminosis, chronic liver diseases, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, allergies, etc.
Medicines using α9α10 nAChR as target for treatment of neuralgia can be delivered administered via intramuscular injection to exert analgesia effects (Vincler, M. Wittenauer, S. Parker, R. Ellison, M. Olivera, B. M. McIntosh, J. M. Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc Natl Acad Sci USA, 2006, 103 (47): 17880-4.), and are more convenient than the currently commercialized ω-CTx MVIIA analgesic, ziconotide. Ziconotide has to be directly delivered to spinal cord via a programmed pump in vivo, so its delivery route is very inconvenient, and the pump is very expensive. At present, it is merely available in developed countries in Europe and America, and can hardly be used in vast developing countries (Kress H G, Simpson K H, Marchettini P, Ver Donck A, Varrassi G. Intrathecal therapy: what has changed with the introduction of ziconotide. Pain Pract. 2009; 9(5):338-47. Burton A W, Deer T R, Wallace M S, Rauck R L, Grigsby E. Considerations and methodology for trialing ziconotide. Pain Physician. 2010; 13(1):23-33. Wallace M S, Rauck R L, Deer T. Ziconotide combination intrathecal therapy: rationale and evidence. Clin J Pain. 2010; 26(7):635-44).
Smoking addiction is caused by nicotine (nicotinamide) in tobacco, and its receptors in body are nicotine acetylcholine receptors (nAChRs) (Azam L, McIntosh J M. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol Sin. 2009; 30(6): 771-783.). Many nAChRs subtypes are not only drug action targets for nicotine addiction, but also drug action targets for drug abuse of morphine, cocaine, etc.
NMDA receptor (N-methyl-D-aspartate receptor) is an important excitatory amino acid receptor in central nervous system, is a ligand-gated ion channel type receptor, and has broad physiological and pharmacological significance. NMDA receptor has significant physiological effects in development of nervous system, such as regulating neuronic survival, regulating structure development of neuronic dendrites and axons, and participating formation of synaptic plasticity; in addition, NMDA receptor also plays a pivotal role in formation of neuronal circuit. At focus of ischemia, NMDA receptor is activated, causing extracellular calcium ion entry, thereby resulting cell death (Twede, V. D., Miljanich, G., Olivera, B. M. & Bulaj, G. Neuroprotective and cardioprotective conopeptides: an emerging class of drug leads. Current opinion in drug discovery & development 2009, 12: 231-239). Studies via rat tests show that NMDA receptor is mainly distributed in central nervous system, such as brain, spinal cord; NMDA receptor is also found in peripheral nervous system, for example, NR3B is mainly expressed in motoneuron, while peripheral NMDA receptors play a very important role in facial muscle pain and edematization.
Studies show that NMDA receptors are very important receptors in processes such as learning, memory, pain, etc., and also attack and treatment targets for many nerve diseases, including refractory pains, drug and alcohol addiction, epilepsy, ischemia, Parkinson's disease, dementia, excitatory neuron death, etc. (Sattler, R. et al. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science (New York, N.Y.) 1999, 284, 1845-1848. Lewis, R. J. & Garcia, M. L. Therapeutic potential of venom peptides. Nature reviews. Drug discovery, 2003, 2: 790-802. Sheng, Z., Liang, Z., Geiger, J. H., Prorok, M. & Castellino, F. J. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B. Neuropharmacology, 2009, 57, 127-136. Meldrum, B. S. The role of glutamate in epilepsy and other CNS disorders. Neurology, 1994, 44:S14-23. Ulas, J. et al. Selective increase of NMDA-sensitive glutamate binding in the striatum of Parkinson's disease, Alzheimer's disease, and mixed Parkinson's disease/Alzheimer's disease patients: an autoradiographic study. The Journal of neuroscience, 1994, 14, 6317-6324. Ozawa, S., Kamiya, H. & Tsuzuki, K. Glutamate receptors in the mammalian central nervous system. Progress in neurobiology, 1998, 54: 581-618. Bisaga, A. & Popik, P. In search of a new pharmacological treatment for drug and alcohol addiction: N-methyl-D-aspartate (NMDA) antagonists. Drug and alcohol dependence, 2000, 59: 1-15).
At present, conotoxin (CTx, conopeptide) generated in venom of conidae, a kind of carnivore mollusc living in tropical ocean, draws a lot of attentions, and is used for systematically studying and developing specific blocking agents for various subtypes of nAChRs.
Conotoxin (conopeptide, CTx) usually a neuropeptide toxin consisting of 7-50 amino acid residues and enriching with cysteine (Cys). Conotoxin can be classified into different gene families according to similarity of precursor protein endoplasmic reticulum targeting sequence and cysteine pattern. So far, all known conotoxins can be classified into 18 superfamilies, i.e., A, B, C, D, S, M, I1, I2, I3, J, L, O1, O2, O3, P, T, V, Y (Kaas Q, Yu R, Jin A H, Dutertre S, Craik D J (2012) ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res 40: D325-330. Sulan Luo, Sean Christensen, Dongting Zhangsun, Yong Wu, Yuanyan Hu, Xiaopeng Zhu, Sandeep Chhabra, Raymond S. Norton, and J. Michael McIntosh. A Novel Inhibitor of α9α10 Nicotinic Acetylcholine Receptors from Conus vexillum Delineates a New Conotoxin Superfamily. PLoS ONE, 8(1): e54648 (1-10), 2013). Conotoxin (conopeptide) can be classified into pharmacological families α, ω, ∥, δ and so on according to receptor target thereof, in which conotoxin of family α (α*-CTx) has function of blocking nicotine acetylcholine receptors (nAChRs); Conantokins as cysteine-free conopeptides have specific function of blocking N-methyl-D-aspartic acid receptor (NMDAR). According to receptor target type, each superfamily of conotoxin can further be classified into α, αA, κA (A-superfamily), ω, δ, κ, μO (O-superfamily), μ, Ψ, κM (M-superfamily), etc. (subtypes).
Conotoxins have special functions of specifically binding various ion channels in animal body. At present, conotoxins have drawn a lot of attentions and are systematically used for studying and developing specific blocking agents for various subtypes of nAChRs.